Our previous experiments, which represent the basis for this proposal, have shown that the cranial neural tube can regulate to form neural crest cells after ablation of the neural folds (Scherson et al. 1993). These experiments suggest that cranial neural tube cells have the potential to become neural crest cells. The goal of this proposal is to examine the cellular and molecular responses underlying the remarkable regulative ability of the neural tube to form neural crest. The proposed experiments will further characterize the effects of ablations to see if similar regulative ability occurs at all axial levels of the neural tube. In addition, we will examine whether other populations of cells, such as the ectodermal placodes, may contribute to neural crest cells when the endogenous neural crest is removed. To examine the molecular nature of the responses underlying this regulative ability, we will characterize the expression pattern after ablation of a variety of marker genes normally found in the dorsal neural tube or neural crest: inducing Slug, Wnt-3a, Pax-3 and some newly isolated genes. The goal is to elucidate which genes are up-regulated after ablation and in what sequence. Having identified candidate genes, we will inhibit their function using antisense oligonucleotides and retrovirally-mediated gene transfer. The effects of such gene "knock-outs" on neural crest formation will help determine which genes are necessary for the regulative response. The experimental design will involve in vivo manipulation by neural fold ablation, coupled with cell marking techniques to identify the origin of neural crest populations, as well as in situ hybridization to look at their gene expression. Specific experiments will examine: 1. genes that are up-regulated in ventral neural tube cells after ablation of the neural folds. 2. regional differences in the regulative response of the neural tube along the neural axis. 3. the importance of inductive interactions between the neural tube and presumptive epidermis in neural crest formation from the ventral neural tube. 4. whether existing neural fold cells inhibit the regulative response of the neural tube. 5. possible contributions of ectodermal placodes to "neural crest" derivatives after neural fold ablation.